We investigated whether transmembrane glucose transport (GT) and glucose phosphorylation in vivo are impaired in NIDD patients and in normal glucose tolerant offspring of two diabetic parents using a novel triple tracer technique developed in our laboratory. Forearm balance and euglycemic insulin clamp studies were performed in 5 groups, including 1)7 healthy controls (C), 2) 5 nondiabetic Mexican-Americans with neither parent diabetic (-FHX), 3) 4 nondiabetic offspring of two NIDD Mexican-American parents, 4) 5 well-controlled NIDD patients (WC), and 5) 5 poorly-controlled NIDD patients (PC). [12C]-mannitol, 3-O-[14-C]-methyl glucose (transportable but not metabolizable) and 3-[3-H]-glucose (transportable and metabolizable) were simultaneously injected into the brachial artery, and their washout curves were measured in deep venous effluent blood. Inward transmembrane GT and intracellular GP were determined by analysis of washout curves using a linear flow compartmental model. Basal forearm blood flow was similar in all groups and was unchanged by hyperinsulinemia. Basal foreamr glucose uptake was similar in the 5 groups, while insulin-stimulated whole body glucose uptake and forearm glucose uptake were higher in the C and -FHX, compated to the +FHX, WC and PC groups. Basal inward GT and GP were similar in all groups. In response to insulin, inward GT increased 2-3 times in the C and -FHX groups and was either unchanged or slightly increased in the +FHX GC and PC groups. Insulin-stimulated intracellular GP also was severely reduced in +FHX, GC and PC groups vs the C and -FHX groups. using pooled data from C, +FHX, -FHX, WC, and PC, a significant positive correlation was found between insulin-stimulated FGU and both GT and FP. Transmembrane GT and intracellular GP in skeletal muscle are impaired in NIDD patients and in the normal glucose tolerant offspring of two diabetic parents and contribute to the insulin resistance.